Abstract: Viruses of the arthropod-borne Flavivirus genus infect over 200 million people worldwide each year, resulting in a significant disease burden in regions where such pathogens are endemic. Although vaccines exist for some of these viruses, there is currently neither effective vaccine nor specific therapy for West Nile virus (WNV), Dengue virus (DNV) or Zika virus (ZIKV). During infection of the host cell, a single viral polyprotein is synthesized from the viral genome. This polyprotein is cleaved into its component proteins by a combination of host cell proteases and a two-component viral protease, encoded in genes NS2B and NS3. The function of this viral protease is essential for virus assembly and replication. The major goal of this proposal is to develop high throughput screening strategies to identify and characterize compounds that allosterically inhibit the function of the ZIKV protease, by preventing the conformational change of the NS2B co-factor. In Specific Aims 1 we will develop novel high-throughput screening assays capable of screening large chemical libraries to identify allosteric inhibitors preventing NS2B from forming correct conformation. In Specific Aim 2 we will perform high throughput screening and evaluate the efficacy of the resulting candidate compounds for their capacity to block protease activity and investigate the allosteric inhibition mechanism. Lead compounds against the protease will be tested for cellular toxicity, reduction of ZIKV titer in cell culture and resistant variants. This study will generate information on flavivirus inhibitor-enzyme interaction at structurally significant sites and potentially lead to novel classes of ZIKV inhibitors.